PROJECT SUMMARY The overwhelming majority of conventional and targeted chemotherapeutics in clinical use or under development rely on engaging apoptotic pathways to elicit tumor cell death. However, resistance to apoptosis- inducing agents is a particularly thorny clinical problem. A novel approach to targeting therapy-resistant cells is to engage cell death mechanisms other than apoptosis to eradicate these malignant subpopulations. The overall goal of the proposed studies is to define the lysosomal-mitochondrial inter-organelle signaling mechanisms underlying tumor cell-specific and programmed necrotic lysosomal cell death (LCD) process induced by a number of drugs. Our lead compound hexamethylene amiloride (HMA), a derivative of a drug that has been employed clinically in the management of blood pressure for over forty-five years, kills differentiated and stem cancer cells independent of tumor type, subtype, or species, but does not efficiently kill normal differentiated cells or stem cells. Moreover, HMA kills cancer cells independent of cell cycle, autophagy engagement, and caspase-dependent apoptosis; indeed, cell death appears to result from drug-induced permeabilization of the lysosomal limiting membrane and subsequent cathepsin-mediated plasma membrane rupture. Our observations indicate that efficient HMA-induced cell death requires the production and action of mitochondrially-produced reactive oxygen species (ROS). Our observations also indicate that HMA induces hallmarks of some of the sphingolipidosis lysosomal storage diseases, including the accumulation of a variety of lipid species that are normally broken down by the lysosome. Notably, lipids such as lactosylceramide and lysophosphatidylcholine that have been demonstrated to act as signaling second messengers in the production of mitochondrial ROS accumulate specifically in tumor cells but not normal cells upon HMA treatment. Our observations point to a model where drug-induced aberrant lipid accumulation and ROS-mediated lysosomal membrane lipid oxidation disrupt lysosomal membrane integrity, allowing cathepsin release and induction of necrotic cell death. To test this model, we will use biochemical, cell biological and metabolomics approaches. In Aim 1 we will assess the contribution of bis(monoacylglycerol)phosphate (BMP), a lysosome resident lipid that is suppressed in tumor relative normal cells and is further suppressed with HMA treatment, in regulating lysosomal membrane stability and cell viability via its ability to activate lysosomal enzymes of the sphingomyelin breakdown pathway. Complementing these studies will be an in-depth analysis of lipidomic and metabolomic changes associated with cellular transformation and LCD-inducing agents. In Aim 2, we will examine lysosomal-mitochondrial signaling events that couple mitochondrial ROS production to dysregulated lysosomal lipid metabolism. These studies will uncover lysosomal targets that will allow future development of novel therapeutic agents that more effectively elicit cancer cell-specific programmed necrotic cell death.